LANKESTERIANA 16(3): 317—327. 2016. doi: http://dx.doi.org/10.15517/lank.v16i3.26649

OSMOPHORE DIVERSITY IN THE CRISTATUM ALLIANCE (: )

Evelyn P. Franken1,2, Ludmila M. Pansarin1 & Emerson R. Pansarin1

1 Biology Department, Faculty of Philosophy, Sciences and Literature of Ribeirão Preto/University of São Paulo - FFCLRP/USP. Av. Bandeirantes, 3900. CEP 14040-901. Ribeirão Preto/SP. Brazil. 2 Author for correspondence: [email protected]

Abstract. Catasetum is a neotropical orchid of 130 species characterized by its unisexual . They are pollinated by male . It is widely know that these bees collect volatile compounds in Catasetum from structures called osmophores. However, there is little information on morpho-anatomy and histochemistry of secretory tissues for this neotropical genus and data are lacking. Based on these arguments members of the Catasetum cristatum alliance, namely C. arietinum, C. ariquemense, C. barbatum, C. carolinianum, C. cristatum, C. lanciferum, C. multifidum, C. multifissum, C. rivularium and C. semicirculatum were analyzed. The labellum of male flowers of this alliance is elongate and with fimbriate margins, possessing two protuberances and a median saccate portion. The labellum of female flowers is galeiform and there is no ornamentation. The entire adaxial surface of the labellum is secretory in both sexes, including the fimbriae of male flowers. The structure of the secretory tissue is similar among species, and they are composed of a simple epidermis and five layers of underlying parenchyma. In most species the epidermis is flat, and cells are elongated in the saccate portion. In C. ariquemense and C. carolinianum the epidermal cells are papillous, while in C. semicirculatum they have convex surface (male flowers). The histochemical analysis detected lipophilic droplets and starch grains inside the secretory cells. All characteristics observed are encountered in secretory tissues with high-energy demand, as is common in osmophores.

Key words: Euglossini bees, floral glands, reward, pollination, volatile compounds Introduction. Nectar is the most common and 2009, Anton et al. 2012), (Curry et al. widespread floral reward among orchids (e.g., van der 1991), (Curry & Stern 1991), Pijl & Dodson 1966). However, a significant number of (Stpiczynska 1993), Gymnadenia (Stpiczynska 2001), species reward their pollinators (i.e., male euglossine Bulbophyllum (Teixeira et al. 2004, Nunes et al. 2014, bees) with volatile compounds. These volatile oils 2015, Kowalkowska et al. 2015), Ophrys (Ascensão et are produced in structures called osmophores (Vogel al. 2005, Francisco & Ascensão 2013), 1990). In Orchidaceae, two types of osmophores have (Pansarin et al. 2008), (Wiemer et al. been reported, namely glandular and epidermal (e. 2009), (Pansarin et al. 2009), Acianthera (de g., Effmert et al. 2006). Osmophores are commonly Melo et al. 2010), (Anton et al. 2012), formed by a single layer of epidermal cells, with uni- Chlorea (Sanguinetti et al. 2012), Vanilla (Pansarin et or multicellular papillae, or several layers of cells. al. 2014), (Pansarin et al. 2014), According to Vogel (1990), osmophores cells have big (Adachi et al. 2015) and Catasetum (Vogel 1963). nuclei, dense cytoplasm, large amount of starch and Catasetum Rich. (Orchidaceae) has approximately high number of mitochondria and other organelles. 130 species distributed mainly through the tropical The majority of these characteristics can be found in region of Central and South America (Romero et al. tissues with high metabolic activity, which is related 2009). The majority of the species occur in the Brazilian to the secretion process (Fahn 2000). Studies about Amazon, where this genus is well represented (Miranda osmophores anatomic structure has been conducted & Lacerda 1992). The species are indistinguishable in a number of unrelated orchid genera, such as in based on the morphology of their vegetative structures. Restrepia (Pridgeon & Stern 1983), Scaphosepalum The flowers are usually unisexual and, occasionally (Pridgeon & Stern 1985), (Stern et al. 1987, hermaphrodite (with no reproductive function). All three Curry et al. 1988, Curry et al. 1991, Pansarin & Amaral flower shapes (staminate, pistillate and hermaphrodite)

Received 13 July 2016; accepted for publication 3 October 2016. First published online: 26 October 2016. Licensed under a Creative Commons Attribution-NonCommercial-No Derivs 3.0 Costa Rica License. 318 LANKESTERIANA have distinct morphology and may occur in the same position to the pollinarium adherence and deposition individual and sometimes in the same (e.g., Dressler 1993). (Romero & Nelson 1986, Romero 1992, Gerlach Since the work of Vogel (1963), none further 2013). Within Catasetum some species alliances are information about flower anatomy of Catasetum recognized based on the morphology of male flowers and has been published. Additionally, no information particularly on the relative position of the staminodes on secretory tissues of species belonging to the (Bicalho & Barros 1988). The “C. cristatum alliance” Catasetum cristatum alliance is available. Based on belongs to the subsection Isoceras, which has parallel these arguments this present work aims to increase or symmetrical antennae (i.e., staminodes). The species the knowledge on flower anatomy of Catasetum, of this group exhibits a strong morphological similarity giving details on the structures related to the resource and new information are required for taxa delimitation production and the tissues involved in the fragrance (Lacerda 1998). secretion of selected species belonging to the “C. For more than fifty years is known that Catasetum cristatum alliance”. species are pollinated by male Euglossini bees (Hymenoptera, Apidae), which collect volatile Materials and methods. Ten species of the compounds at the flowers (Vogel 1963). In this case, Catasetum cristatum alliance were analyzed (Tab. 1). the fragrance compounds are volatilized and act as an The specimens were collected in natural areas from attractant and a resource for male euglossine bees that Brazil or bought from commercial orchids cultivators collect them with a hard hair structure present on their and maintained in the Orchidarium LBMBP of the anterior legs and are stored into the hind tibiae (Eltz et Department of Biology, FFCLRP, São Paulo University, al. 1999). These fragrances consist mainly on a variety in the city of Ribeirão Preto (approx. 21º10’S, 47º48’W; of terpenoids and aromatics that sometimes possess 546 m a.s.l.), state of São Paulo, southeastern Brazil. a species-specific composition (Hills et al. 1972, In some cases more than one individual of a same Gerlach & Schill 1991). During fragrance collection, locality were potted with Pinus bark in a single pot. bees incidentally contact the antennae of staminate The vouchers material (living ) was deposited at (male) flowers and the pollinarium is deposited on the the orchidarium of the Molecular Biology and Plants ’s body (mainly on the scutum). When visiting a Biosystematics Lab (Tab.1). pistillate (female) flower, the pollinia deposition in The morphological features of fresh flowers of the stigmatic cleft can occur (Dodson 1962, Dressler Catasetum cristatum alliance were examined under 1968). Flower morphology and osmophore disposition a binocular stereomicroscope. To determine the on the labellum may guide the pollinator for a correct osmophores location in each species, flowers were

Table 1. Species analyzed, locality (states of Brazil), flower sex (Male/Female), number of specimens analyzed (N) and reference number (RN) at LBMBP Orchidarium.

Species Provenance Flower sex N RN Catasetum arietinum Miranda & Lacerda PE M 1 844 Catasetum ariquemense Miranda & Lacerda RO M 1 924

Catasetum barbatum (Lindl.) Lindl. AM M 1 952 Catasetum carolinianum Miranda & Lacerda GO M 1 946 Catasetum cristatum Lindl. co* M 1 990 Catasetum lanciferum Lindl. MG M 3 943 Catasetum multifidum Miranda PA M and F 2 815, 818 Catasetum multifissum Senghas RO M 1 916 Catasetum rivularium Barb.Rodr. AM M 1 955 Catasetum semicirculatum Miranda RO M 1 917 * Commercial orchidary.

LANKESTERIANA 16(3). 2016. © Universidad de Costa Rica, 2016. Franken et al. — Osmophores in C. cristatum alliance 319 collected during the fragrance production (2 to 5 protuberances are composed by a single tooth, three days after flower opening in daylight period) from or numerous teeth. In some cases, the apical callus (C. each specimen. The flowers were immersed in 0.1% barbatum and C. cristatum) or both calli (C. rivularium) (w/v) aqueous neutral red for 1-24 h (Vogel 1962). split in a cluster of fimbriae. In C. multifidum and Additionally, they were longitudinally sectioned with C. multifissum the apical portion of the labellum is a razor blade and immersed in a Lugol 1% solution tridentate and branched. In the rest of the labellum there for 1-10 min to localize tissue containing starch. The is no evident ornamentation and the surface is smooth. histochemical tests were made on transversal section of In female flowers ofC. multifidum, the secretory surface the labella stained with Sudan IV (Pearse 1985) to detect is smooth and the secretory tissue is homogeneously total lipids, and Lugol 1% (Hinchman 1973) for starch distributed. grains. To characterize the anatomical structure, flowers The structure of the secretory tissue is similar among of each species were fixed in formalin-acetic acid- species belonging to the C. cristatum alliance. The alcohol (FAA 50%) for 24 h (Johansen 1940) and stored osmophore has an epidermis and up to five subepidermal in 70% ethanol. The fixed material was dehydrated in parenchymal layers. This structure is followed by ethanol series and embedded in Historesin (Leica). It vascular bundles, a non-secreting parenchyma and an was sectioned at a thickness of 10-12 µm using a rotary abaxial epidermis (Fig. 4A). Variation in the epidermal microtome. The sections were stained with Toluidine cells morphology of male flowers was observed. In the Blue (O’Brien et al. 1964). Observations were carried majority of the studied species (i.e. C. arietinum, C. by using a light microscope Leica DM500. Images with barbatum, C. cristatum, C. lanciferum, C. rivularium a minimum of 600 dpi resolution were captured with a C. multifidum, and C. multifissum) the epidermal digital camera Leica ICC50 attached to the microscope. cells surface are tabular (flat) and anticlinal walls are The terminology on morphology of secretory cells tetragonal (on the calluses) to tetragonal-elongated (on follows Koch et al. (2008). the saccate portion) (Figs. 4B-C). In C. ariquemense the cells surface is papillous (on the calluses) to tabular Results. The flower morphology is similar among with convex hunches (on the saccate portion) (Fig. 4D). species of the Catasetum cristatum alliance. The male In C. carolinianum the cells surface is papillous (on the flowers are resupinate, and the sepals and petals are calluses) to convex (on the saccate portion). Catasetum membranous, green and generally brown-spotted. semicirculatum has cells with a convex surface (Fig. Sepals are oblong-lanceolate, concave and with acute 4E). In female flowers of C. multifidum, the epidermis apex. The dorsal sepal is erect, while the lateral sepals shows a surface with tabular cells, where its anticlinal are reflexed. Petals are oblong-lanceolate, convex, walls are tetragonal (Figs. 4F). A thick cuticle covers the erect, and with an acute apex. The labellum is elongate, epidermis of both male and female flowers. This cuticle, fleshy, with a saccate cavity in the middle portion, and because of his hydrophobic characteristic, seems to with fimbriate margins (Figs. 1A-S). The adaxial surface prevent the neutral red staining of osmophores from all of the labellum possesses two protuberances, named species analyzed. The subepidermal parenchyma cells basal and apical calluses. The female flowers are non- of both male and female flowers are anisodiametric resupinate and the sepals and petals are membranous, and bigger than epidermal cells. The non-secreting strongly reflexed, and entirely green. The labellum is parenchymal cells of female flowers have large galeiform, and very fleshy (Fig. 2). Protuberances are intercellular spaces, contrasting to male flowers with absent and the surface is longitudinally ribbed due the more compact tissues. presence of prominent vascular bundles. The fimbriate margins of male flowers seem to In all species analyzed, the secretory tissue occurs on be specialized structures for volatile emission, being the adaxial surface of the labellum of male and female composed almost entirelly by secretory cells. The flowers. The osmophore covers the entire surface of epidermis are partially (C. arietinum, C. barbatum, C. the labellum, extending to the margins of this structure, multifidum, C. multifissum; Fig. 5A-B) or totally (C. including the fimbriae (Figs. 3A-F). In male flowers, cristatum, C. rivularium, C. semicirculatum) composed the secretory tissue is very evident on both calli. These by flat cells or entirely composed by papillous cells

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(C. ariquemense, C. carolinianum, C. lanciferum; Fig. 5C). Internally, the fimbriae possess thin vascular bundles (Fig. 5D). In all species studied (male and female flowers) both epidermal and parenchymal cells of the secretory tissue are rich in oil droplets, which are stained with Sudan IV, confirming the lipophilic nature of the secretion in all the species analyzed (Figs. 6A-C). A large amount of starch grains is detected in the subepidermal parenchyma, but not in the epidermis (Figs. 6D). In the beginning of the scent production, the grains are aggregated and filled all the space inside the cells. After a few days those reserves are used, Figure 2. Female flower ofCatasetum multifidum. Note that letting remain only small grains. the female flowers are non-resupinate. Scale bar = 1 cm. Discussion. Catasetum offer floral fragrances Sievekingia and Stanhopea) have their osmophores as a reward, attracting male euglossine bees as pattern described in detail. Cycnoches chlorochilon pollinators. In fact, its flowers are completely adapted has the most similar structure of the secretory tissue to the euglossinophilous pollination syndrome. Their comparing with what we recorded in members of flowers possess big osmophores, where the chemical the C. cristatum alliance, but the secretory tissue is compounds produced inside the secretory cells localized only in an egg-shaped cavity of the labellum volatize to attract male bees from long distances. To (Anton et al. 2012). In Cirrhaea, the secretory tissue do that, the secretory tissue covers the entire adaxial is localized in a midlobe protuberance, and extends to surface of the labellum. Vogel (1990), describing the the basal portion of the inner side of the lateral lobes male flowers (labellum with galeiform morphology) of the labellum (Pansarin et al. 2006, Pansarin et al. of C. macrocarpum Rich. ex Kunth, C. fimbriatum 2014). The flowers ofStanhopea and Gongora bufonia (Morren) Lindl. and C. maculatum Kunth, clarified are very specialized, presenting a differentiated the anatomy and the nature of the resource produced. hypochile in the labellum, region where the secretory He described the labellar tissues according to its tissue is located (Stern et al. 1987, Curry et al. function in the following order: emission layer 1991, Pansarin & Amaral 2009, Anton et al. 2012, (adaxial epidermis), plasma-rich production layer Adachi et al. 2015). In Sievekingia, the labellum is (secretory parenchyma), horizon of veins (vascular less differentiated than Stanhopea and Gongora, bundles), parenchyma (not-secretory parenchyma) presenting the osmophore in the base of a shallow and outer epidermis (abaxial epidermis). Warford pouch behind the callus (Curry et al. 1991). Grobya and Harrell (1996) only indicated the location of amherstiae and Cyrtopodium polyphyllum are closely the osmophores in male and female flowers of related to Catasetum (e.g., Freudenstein et al. 2004), C. pendulum Dodson using a neutral red staining but belong to another pollination syndromes. G. method. The same osmophore pattern described amherstiae (Catasetinae) is pollinated by females of by Vogel (1990) has already been documented for Paratetrapedia fervida Smith (Anthophoridae) bees another euglossinophilous species, but not at the same that collect edible oil from the elaiophores (Pansarin size and complexity, continually covering plain and et al. 2009). This species produce volatile compounds sacciform areas, calluses and fimbriae of the labellum. only to attract effective pollinators to the flowers, and Only one Catasetinae (Cycnoches chlorochilon) and its osmophore is epidermal and located on abaxial some genera of (Cirrhaea, Gongora, surface of the labellum. Cyrtopodium polyphyllum

Left, Figure 1. Diversity in male flowers of the Catasetum cristatum alliance. A - B. C. arietinum. C - D. C. ariquemense (note the saccate cavity - SC). E - F. C. lanciferum. G - H. C. carolinianum. I - J. C. semicirculatum. K - L. C. multifi- dum. M - N. C. multifissum. O - P. C. rivularium. Q - R. C. cristatum. S. C. barbatum. Scale bars: A - S = 1 cm.

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Figure 3. Arrangement of the secretory tissue (based on tests with Lugol 1%), in the labellum of male flowers of the Cata- setum cristatum alliance. A. C. arietinum. B. C. ariquemense. C. C. cristatum. D. C. carolinianum. E. C. multifidum. F. C. rivularium. Note that in all species analyzed the secretory tissue is stained in black (arrows). Scale bars: A - F = 1 cm. (Cyrtopodiinae) is pollinated by deceit by Centridini These variations have an evolutionary relevance, bees and produce scent as attractant (Pansarin et al. were the selective pressure seems to have favoured 2008). The osmophores are multicellular protrusions, the development of more elaborate osmophores with more evident on the callus of the labellum. papillae, rugae and trichomes. In Cirrhaea spp. the The species of C. cristatum alliance show some osmophores do not show interspecific variation in variation in epidermal cell morphology. The meaning epidermal cells (Pansarin et al. 2014). All the seven of these modifications can have several explanations. species analyzed by these authors have epidermal Whitney et al. (2011), comparing flowers with flat osmophores with tabular cells covered by a thin cuticle. and papillous cells, showed that presence of conical However, comparative or evolutionary studies of epidermal cells in petals enhance pollination success. secretory structures in different species of the same genus This cells shape affect significantly flower colour, are still very scarce. The subepidermal parenchymal flower wettability and the tactile handling of the flower cells of C. cristatum alliance seem to follow the same by pollinators. However, have limited effect on floral morphology and size pattern, corroborating with Stern temperature and might have an indirect effect on scent et al. (1987). They observed that the morphology dispersal. Interspecific variation in epidermal cells and ultrastructure of all cells within this region was was reported on Stanhopea by Curry et al. (1991). essentially the same in Stanhopea wardii and S. oculata.

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Figure 4. Labellum anatomy in the Catasetum cristatum alliance (stained with Toluidine blue). A. Transversal section of female flower labellum of C. multifidum; Note the secretory tissue of the adaxial surface (Ad), and the abaxial surface (Ab), with a thin epidermis and the parenchymal cells with large intercellular spaces. B. Epidermis on the apical callus of male flowers of C. lanciferum. C. Epidermis on the saccate portion of male flowers of C. lanciferum. D. Epidermis on the basal callus of male flowers ofC. ariquemense. E. Epidermis of the basal callus of male flowers ofC. semicirculatum. F. Epidermis of female flowers of C. multifidum; Note the thick cuticle (arrows). Scale bars: A = 200 µm; B - F = 50 µm.

The labellum morphology and anatomical mechanism depends critically on the precision of features have an important function in pollination pollinarium emplacement on the bee (e.g., van der mechanism. The effectiveness of the pollination Pijl & Dodson 1966). To do that, the distribution of

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Figure 5. Fimbriae of male flowers of theCatasetum cristatum alliance. A. C. arietinum; Note the epidermis with papillous cells in the apex (arrow). B. Fimbriae apex of C. multifidum. C. C. lanciferum; Note the epidermis with papillous cells (arrow). D. Vascular bundle internally on the fimbriae ofC. multifidum. Scale bars: B and D = 50 µm. the secretory tissue must guide the bee below the reception on the stigma is passive, and the secretory column. According to Dodson (1962), there is a tissue distribution in female flowers stimulates the relation between the position of the antennae (the bee to enter in the galeiform labellum for resource trigger of pollinarium ejection mechanism) and the collection. The pollinia is deposited on the stigmatic secretory tissue. Dodson (1962), cited for Catasetum cleft of the column when the bee finish the collection macroglossum: “the antennae are located just over and the labellum interior (e.g., Dodson 1962). the deepest point of the saccate labellum where the The female flowers have longer longevity (e.g., strongest odour is emitted”. In addition, the thicker Milet-Pinheiro et al. 2015). However, we have not secretory tissue of basal callus in male flowers has noticed any relevant modification in the secretory the function to produce resource for collection, tissue of female flowers when compared to male which results in a correct bee positioning below the flowers. column and allow the contact with the antennae. The C. cristatum alliance have the most complex lip According to Vogel (1990), this resource does not morphology among Catasetum. The labellum is open stay available for a long time, and evaporate rapid. and shows extraordinary structures, such as calluses The fragrance produced in thinner secretory tissue, in and fimbriae. The three species analyzed by Vogel the rest of lip areas, not seem to produce a collectible (1963, 1990) have more simple labellum morphology. amount of resource. It probably have only attraction However, both species (analyzed by him and us) function for pollinators in these areas. The pollinia show similar structure of the secretory tissue. Finally,

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Figure 6. Histochemical tests on the labellum of male flowers of the Catasetum cristatum alliance. A. Lipids droplets in C. rivularium callus (arrows) - stained with Sudan IV. B. Lipids droplets in C. carolinianum saccate portion (arrows) - stained with Sudan IV. C. Lipids droplets into a fimbriae of C. barbatum (arrows) - stained with Sudan IV. D. Starch grains in C. semicirculatum - stained with Lugol 1%. Scale bars: A - D = 50µm. we were able to increase knowledge to the genus, Literature cited highlighting the role of the osmophores in pollination Adachi, S. A., Machado, S. R. & Guimarães, E. (2015). mechanism. The characteristics described here can Structural and ultrastructural characterization of also be useful in the of this neotropical and the floral lip in Gongora bufonia (Orchidaceae): diverse orchid genus. understanding the slip-and-fall pollination machanism. Botany, 93, 759-768. Anton, S., Kaminska, M. & Stpiczynska, M. (2012). Comparative structure of the osmophores in the flower of Stanhopea graveolens Lindley and Acknowledgements. The authors thanks Silvia R. M. Cycnoches chlorochilon Klotzsch (Orchidaceae). Acta Pedro for help us with the slides preparation. This study Agrobotanica, 65, 11-22. is part of the PhD Thesis of EPF in the Post-graduation Ascensão, L., Francisco, A., Cotrim, H. & Pais, M. S. Program in Comparative Biology at the FFCLRP-USP. The (2005). Comparative structure of the labellum in Ophrys first author received a scholarship from State of Amazonas fusca and O. lutea (Orchidaceae). American Journal of Research Foundation (FAPEAM/SECTI/Governo do Botany, 92, 1059-1067. Estado do Amazonas). This research was supported by the Bicalho, H. D. & Barros, F. (1988). On the taxonomy of State of São Paulo Research Foundation (FAPESP; Grant Catasetum subsection Isoceras. Lindleyana, 3, 87-92. 2014/14969-6). Curry, K. J., Stern, W. L. & Mcdowell, L. M. (1988).

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